PROJECT SUMMARY/ABSTRACT Breast cancer prognosis varies among subtypes, and while molecularly targeted therapies have improved outcomes for estrogen receptor (ER)-expressing and HER2-overexpressing tumors, triple negative breast cancers lacking these molecular targets, most of which also belong to the basal-like molecularly-defined subtype, have a poorer prognosis. Matrix metalloproteinase-9 (MMP-9) has been implicated as a driver of breast cancer progression, metastasis, and angiogenesis, and offers a promising target for therapy, particularly for the basal-like breast cancer subtype. In earlier clinical trials of small molecule MMP inhibitors, lack of selectivity of the inhibitors led to poor efficacy and serious side effects. We hypothesize that better results may be attained using MMP-9 inhibitors derived from a natural protein inhibitor of MMP-9, tissue inhibitor of metalloproteinases-1 (TIMP-1). In this project, we propose to employ a variety of complementary methods to engineer TIMP-1 as a therapeutic protein for effective in vivo targeting of MMP-9 activity, and to evaluate the therapeutic potential of resulting engineered TIMP-1 variants. In Aim 1, using insights from our recently reported crystal structures of MMP/TIMP complexes, we will take a novel directed evolution approach to enhancing the binding selectivity of TIMP-1 for MMP-9. In Aim 2, we will define TIMP-1 epitopes that are essential for MMP-independent signaling, and determine the feasibility of mutating TIMP-1 to eliminate these off-target activities while preserving MMP-9 and proMMP-9 affinity. In Aim 3, we will use our recently- developed methodology to PEGylate recombinant TIMP-1 variants, improving their pharmacokinetic profile, and evaluate these TIMP-1-based therapeutics in biologically relevant preclinical models of basal-like breast cancer. Our proposal is both conceptually and technically innovative in the combination of approaches toward generating novel MMP-9-targeted protein therapeutics. The proposed research is highly significant because it has substantial potential to develop an entirely new class of drugs for breast cancer, targeting a key driver of invasion, metastasis, and angiogenesis. We expect selective MMP-9 inhibitors to be particularly useful for treating HER2-negative, endocrine nonresponsive cancers for which other targeted therapies are ineffective.